La recherche a été soutenue par la Fondation de recherche Harry Winston.

1. Préparer une solution fraîche Solutions de travail <ol><li> SCT: 0,05 M Tris Buffered Saline, pH 7,8 (TBS). Préparer 60 ml TBS / microplaque. </li><li> EPI: l&#39;élastase pancréatique porcine, de type 1 (PPE) est fourni sous forme de suspension. Bien agiter et diluer EPI SCT à environ 0,01 U / ml. Préparer 6 ml de solution de travail / microplaque). </li><li> SA 3 NA: succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA 3 NA). Préparer 0,1 M de stock SA solution 3 NA dans le diméthylsulfoxyde (DMSO) et utiliser immédiatement ou conserver à -20 ° C. Préparer la solution de travail 6ml / microplaque par dilution de stock SA solution 3 NA 1/100 dans du TBS. </li><li> Bleu de Coomassie: Préparer une solution stock de 10% de bleu de Coomassie R-250 (Bleu de Coomassie) dans du TBS et utiliser immédiatement ou conserver à -20 ° C. Préparer 6 ml de solution de travail en diluant la solution stock de 1/1000 dans du TBS. </li></ol> 2. Préparer la salive <ol&gt; <li> Prélèvement de salive: En position debout, de stimuler l&#39;exsudation du sérum dans la salive en plaçant un sialogogue tels que l&#39;acide citrique ou de chewing-gum dans la bouche. Une goutte de citron sans sucre est commode pour les patients VIH-1, qui sont souvent édentés et incapable de mâcher de la gomme. Avalez le cas échéant pour la première fois 3 min, et de recueillir la salive de toute la bouche dans une tasse plafonné pour le prochain 3 min. Cela permettra d&#39;environ 2 ml de salive. Utiliser immédiatement ou conserver à -80 ° C. </li><li> MANIPULATION DE SALIVE: Si congelée spécimen dégel, à 37 ° C et le lieu sur la glace. Stockez spécimen frais ou décongelés à 2-4 ° C ne dépasse pas 3 jours. Avez salive pas de tourbillon, et d&#39;éviter de pipetage mucine qui ne contient pas d&#39;exsudat de sérum. Utilisez les précautions standard lors de la manipulation de salive <a href="http://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdf" target="_blank">http://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdf</a> . </li></ol> 3. Microplaques Set-up <ol><li><strong&gt; TAMPON: Les mesures sont effectuées en triple exemplaire. Pour chaque puits d&#39;une plaque de microtitration est ajouté 130 pi SCT. </li><li> SPECIMENS: Pour chaque puits de test, on ajoute 20 ul de salive pour un volume final de 150 pl / puits. Mélanger à la pipette sans introduire de bulles. </li><li> COMMANDES: Le contrôle positif contient PPE, mais pas de la salive. Le contrôle négatif contient de la salive, mais pas PPE.To le contrôle positif est bien ajouté 20 uL du SCT. Pour le contrôle négatif est bien ajouté 20 uL de salive. </li><li> INCUBATION EPI: Agiter la solution PPE fonctionne bien. Ajouter 50 uL solution de travail PPE à chaque puits à l&#39;exclusion des puits témoins négatifs. Pour les puits de contrôle négatif, ajouter 50 ul du SCT. Incuber 10 min à 23 ° C. </li><li> INCUBATION DU SUBSTRAT: Ajouter 50 uL SA solution 3 NA travaille à chaque puits. Incuber 45 min à 23 ° C. </li><li> Bleu de Coomassie:. Ajouter 50 ul bleu de Coomassie solution de travail &lt;/ Li&gt; <li> LECTURE: Lire l&#39;absorbance dans un lecteur de microplaques, un 405 nm pour détecter SA 3 clivage NA qui représente activement α 1 PI et A 595 nm pour détecter au bleu de Coomassie qui représente la teneur en protéines. </li></ol> 4. Calculer α 1 Indice PI <ol><li> NORMALIZE LA MOYENNE A 405 nm et un nm 595: Pour chaque trois exemplaires, calculer la moyenne. Soustraire la moyenne du contrôle négatif de la moyenne des échantillons. Afin de minimiser la plaque à la plaque de variation, de normaliser les moyennes en divisant chaque moyenne des échantillons par la moyenne de contrôle positif comme suit: </li></ol><img alt="L&#39;équation 1" src="/files/ftp_upload/3999/3999eq1.jpg" /> <ol start="2"><li> CALCUL DE LA α 1 PI INEX: Diviser le normalisé A 405 nm par les normalisés A 595 nm: </li></ol> 5. Les résultats représentatifs Pour déterminer si l&#39;α-test peut être adapté pour être utilisé comme un test de dépistage au point de soins pour surveiller la numération des CD4 dans les pays endémiques, des régions à ressources limitées, la salive stimulée a été recueillies auprès de 20 femmes et 11 hommes VIH-1 clinique des sujets traitant pour des soins de routine au Cameroun. Concorde avec les observations préliminaires au cours du développement de l&#39;α-test à l&#39;aide de salive recueillis à New York, le α 1 Indice PI dans la salive recueillie au Cameroun en corrélation avec le nombre de CD4 (r 2 = 0,91, p &lt;0,0001. N = 31). La relation a été définie par une courbe sigmoïde 3-paramètre qui est compatible avec une relation dose-réponse (figure 2A). La confiance de 95% et des intervalles de prédiction sont représentées par des lignes bleues et rouges, respectivement (figure 2B). Dansune étude précédente, nous avons déterminé que les cellules T CD4 présentent vélo sinusoïdale avec une périodicité de 23 ± 3,5 jours dans le VIH-1 sujets et chez les sujets atteints de la version héritée de α carence en 1 IP (PIZZ) 3. Présentée dans le présent est un exemple représentatif de l&#39;ordinateur généré par analyse de la courbe sinusoïdale de CD4 vélo dans un non-VIH-1 présentant 27 témoins sains périodicité jour (figure 3A). Générée par ordinateur d&#39;analyse courbe sinusoïdale des cellules CD4 + changements de cellules T chez 6 sujets a donné des valeurs de crête à crête d&#39;amplitude et de l&#39;axe d&#39;oscillation 3. Comme on pouvait s&#39;y attendre, l&#39;axe d&#39;oscillation a été corrélée avec une amplitude (r 2 = 0,96, p = 0,009, n = 6) (figure 3B). Chez les patients seront CD4 bas T + nombre de cellules, les changements cycliques de la numération de lymphocytes T CD4 étaient de petite taille, et chez les patients atteints de CD4 + élevé de lymphocytes T, les changements cycliques étaient grandes. Parce que ledroite de régression montre la figure 2 estime l&#39;axe d&#39;oscillation et de l&#39;axe d&#39;oscillation est corrélée avec une amplitude, l&#39;amplitude peut être calculée pour la ligne de régression estimant ainsi dans quelle mesure ci-dessus et ci-dessous les CD4 + de la ligne de régression des cellules T on peut s&#39;attendre à varier en raison de vélo (figure 3C). Superposer l&#39;amplitude calculée à la figure 3C et l&#39;intervalle de confiance calculé à la figure 2B, on a constaté que l&#39;amplitude (ligne verte) se situe dans les 95% l&#39;intervalle de confiance (ligne bleue) (figure 3D). <img alt="Figure 1" src="/files/ftp_upload/3999/3999fig1.jpg" /> Figure 1. Résumé de la procédure: l&#39;acide citrique dans la bouche de la Place et de recueillir la salive. Ajouter la salive dilués dans les puits d&#39;une microplaque en triple exemplaire. Ajouter EPI à chaque puits et incuber pendant 15 min à 23 ° C. Ajouter le substrat PPE à chaque puits et incuber pendant 45 min à 23 ° C. Ajouter au bleu de Coomassie et lire à 405 nm pour détecter l&#39;activité de protection individuelle et à 595 nm pour détecter la protéine. <img alt="Figure 2" src="/files/ftp_upload/3999/3999fig2.jpg" /> Figure 2. Comparaison de l&#39;α-test avec la méthode d&#39;énumération CD4 standard. A) Le α-test a été réalisé par la quantification de la α 1 Indice PI dans la salive la bouche stimulé l&#39;ensemble. La numération des CD4 ont été réalisées par un laboratoire médical indépendant utilisant la cytométrie de flux standard. Sang pour la cytométrie de flux et de salive ont été prélevés lors de la visite même clinique. La relation entre l&#39;α 1 Indice PI et la numération des CD4 a été définie par une courbe sigmoïde à 3 paramètres (r 2 = 0,91, p &lt;0,0001, n = 31). B) La confiance de 95% et des intervalles de prédiction sont représentées en bleu et rouge lignes, respectivement. <img alt="Figure 3" src="/files/ftp_upload/3999/3999fig3.jpg" /> Figure 3. Précision de calcul CD4 contants en utilisant le Index.A α1PI) cellules T CD4 + exposé vélo sinusoïdale avec 27 périodicité jour dans un contrôle non-VIH-1 en bonne santé. L&#39;axe d&#39;oscillation est représenté (pointillé rouge). B) Computer analyse généré onde sinusoïdale de CD4 des lymphocytes T du vélo a été utilisé pour calculer l&#39;amplitude de crête à crête et de l&#39;axe d&#39;oscillation dans 4 sujets VIH-1, 1 non-VIH -1 Pizz sujet, et le non-VIH-1 de contrôle sain représenté dans la partie A. La relation entre T CD4 + amplitude cellule et axe d&#39;oscillation a été définie par le 3-paramètre courbe sigmoïde (r 2 = 0,96, p = 0,009, n = 6). C) La relation sigmoïde entre l&#39;amplitude et l&#39;axe d&#39;oscillation provenant en partie B) a été utilisée pour calculer l&#39;amplitude attendue (lignes vertes) de points le long de la ligne de régression (ligne noire) de la figure 2. D) L&#39;amplitude attendue intervalle (lignes vertes) était significativement différente de l&#39;intervalle de confiance 95% (lignes bleues) comme déterminé par le test de Mann-Whitney somme des rangs (basée sur une comparaison de la différence moyenne entre l&#39;intervalle de confiance de 95% et la ligne de régression (63 cellules / uL) et la différence moyenne entre la ligne de l&#39;amplitude et la droite de régression (37 cellules / pl); p = 0,001).

<ol>
	<li>Friedlander, Y., Kark, J. D., Eisenberg, S., Stein, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+LDL-cholesterol+from+total+cholesterol,+triglyceride+and+HDL-cholesterol:+a+comparison+of+methods+in+the+Jerusalem+Lipid+Research+Clinic+Prevalence+Study.">Calculation of LDL-cholesterol from total cholesterol, triglyceride and HDL-cholesterol: a comparison of methods in the Jerusalem Lipid Research Clinic Prevalence Study.</a> Isr. J. Med. 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Pas de conflits d&#39;intérêt déclarés.

Sérum non dilué contient médiane 36 uM α 1 PI et 3,6 uM α 2-macroglobuline (α 2 M) une différence de 10 fois 8. Les deux protéines compétition pour la liaison aux EPI. Ces deux caractéristiques, la concentration et l&#39;affinité EPI, ont été exploitées pour développer une méthode qui est capable de mesurer précisément α 1 PI dans le sérum normal en présence de concurrence α 2 M 8. Cette méthode permet de détecter aussi ...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Sialogogue, 0,05 M d&#39;acide citrique </td><td> Bonbons sans sucre eda, 4900, rue arrière N.20th, Philadelphia, PA 19144 </td><td> Gouttes de citron sans sucre </td></tr><tr><td> Élastase pancréatique porcine, de type 1 (CE 3.4.21.36) </td><td> Sigma-Aldrich, St. Louis, MO 63103 </td><td> E1250 </td></tr><tr><td> Succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide </td><td> Sigma-Aldrich </td><td> S4760 </td></tr><tr><td> Bleu de Coomassie R-250 </td><td> Sigma-Aldrich </td><td> B7920 </td></tr><tr><td> La culture de tissus traités, 96 microplaques ainsi </td><td> Becton Dickinson, Franklin Lakes, NJ 07417-1886 </td><td> 35-3072 </td></tr><tr><td> Lecteur de microplaques avec des filtres pour 405 nm et 595 nm </td><td> MTX Systems Lab, 8456 Tyco Road, Building D, Vienna, Virginia 22182 </td><td> Dynex P97277 </td></tr></tbody></table>

the &alpha;-test: rapid cell-free cd4 enumeration using whole saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

Watch this Scientific Journal Video about The Î±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva at JoVE.com

The &amp;#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

Une méthode de numération des CD4, l&#39;α-test, est décrite qui utilise la salive ensemble, de fournir rapides et précises des taux de CD4. Les sous-α essai coûts et élimine la nécessité d&#39;une formation technique, des réactifs coûteux tels que les anticorps monoclonaux, l&#39;instrumentation, de réfrigération, le transport des échantillons, ainsi que de collecte et de traitement du sang.

Le α-test: Rapid portable sans énumération des CD4 utilisant la salive entier

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

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Research

JoVE Journal

Medicine

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

13.7K vues.  Weill Cornell Medical College. Une méthode de numération des CD4, l'α-test, est décrite qui utilise la salive ensemble, de fournir rapides et précises des taux de CD4. Les sous-α essai coûts et élimine la nécessité d'une formation technique, des réactifs coûteux tels que les anticorps monoclonaux, l'instrumentation, de réfrigération, le transport des échantillons, ainsi que de collecte et de traitement du sang.

Vidéo: The &#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

Mesure précise et simple de la cytokine pro-inflammatoire IL-1β en utilisant un dosage de sang total Stimulation

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

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Médecine

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

Rapid Point-of-Care Test de l&#39;efficacité anticoagulante énoxaparine dans le sang total

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4 may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.
Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7 and pre-digestion with trypsin, which makes it difficult for clinical use. 
To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo from various microenvironments in animals in a dynamic and minimally invasive manner 8-11. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.

Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.

Échantillonnage humaines Peptidome salive autochtones l&#39;aide d&#39;une sonde d&#39;ultrafiltration Lollipop-Like: Simplifier et améliorer la détection des peptides pour spectrométrie de masse clinique

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Collecte et d&#39;extraction de l&#39;ADN de la salive pour le séquençage de prochaine génération

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Fractionnement rapide et isolement de composants de sang total dans les échantillons obtenus à partir d&#39;un milieu communautaire

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

Évaluation des propriétés anticoagulantes et anti-inflammatoires de cellules endothéliales à l’aide de la Culture cellulaire 3D et sang total Non-anticoagulant

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and no new nephrons are formed post-birth. Nephron number refers to the total number of nephrons measured at any point in time post-birth. Both genetic and environmental factors influence both nephron endowment and number. Understanding how specific genes or factors influence the process of nephrogenesis and nephron loss or demise is important as individuals with lower nephron endowment or number are thought to be at a higher risk of developing renal or cardiovascular disease. Understanding how environmental exposures over the course of a person's lifetime affects nephron number will also be vital in determining future disease risk. Thus, the ability to assess whole kidney nephron number quickly and reliably is a basic experimental requirement to better understand mechanisms that contribute to or promote nephrogenesis or nephron loss. Here, we describe the acid maceration method for the estimation of whole kidney nephron number based on the procedure described by Damadian, Shawayri, and Bricker, with slight modifications. The acid maceration method provides fast and reliable estimates of nephron number (as assessed by counting glomeruli) that are within 5% of those determined using more advanced, albeit expensive, methods such as magnetic resonance imaging. Moreover, the acid maceration method is an excellent high-throughput method to assess nephron number in large numbers of samples or experimental conditions.

Estimates of whole kidney nephron number are important clinically and experimentally, as there is an inverse association between nephron number and an enhanced risk of renal and cardiovascular disease.Herein, the use of the acid maceration method, which provides fast and reliable estimates of whole kidney nephron number, is demonstrated.

Estimation du nombre de néphron dans le rein entier à l’aide de la méthode de macération acide

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

Évaluation rapide de la toxicité des composés chimiques à l'aide d'embryons de poisson zèbre

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G&#39;, G&#34;, G*, and tan &#948;) and gel point characteristics (&#947;c&#160;and &#963;c) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is &#60;10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more &#34;liquid-like&#34; behavior overall (e.g., higher tan &#948;). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.

The viscoelastic properties of mucus play a critical role in mucociliary clearance. However, traditional mucus rheological techniques require complex and time-consuming approaches. This study provides a detailed protocol for the use of a benchtop rheometer that can rapidly and reliably perform viscoelastic measurements.

Caractérisation viscoélastique rapide du mucus des voies respiratoires à l’aide d’un rhéomètre de paillasse

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., ...

Whole-Kidney Three-Dimensional Staining with CUBIC

Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood vessels, proximal tubules, collecting ducts, glomeruli, and sympathetic nerves in the kidney due to the lack of three-dimensional (3D) information. Optical clearing is a good strategy to overcome this big hurdle. Multiple cells in a whole organ can be analyzed at single-cell resolution by combining tissue clearing and 3D imaging technique. However, cell labeling methods for whole-organ imaging remain underdeveloped. In particular, whole-mount organ staining is challenging because of the difficulty in antibody penetration. The present protocol developed a whole-mount mouse kidney staining for 3D imaging with the CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) tissue clearing method. The protocol has enabled visualizing renal sympathetic denervation after ischemia-reperfusion injury and glomerulomegaly in the early stage of diabetic kidney disease from a comprehensive viewpoint. Thus, this technique can lead to new discoveries in kidney research by providing a macroscopic perspective.

The present protocol describes a tissue clearing method and whole-mount immunofluorescent staining for three-dimensional (3D) kidney imaging. This technique can offer macroscopic perspectives in kidney pathology, leading to new biological discoveries.